Novel all-trans polyene amphoteric macrolide

ABSTRACT

The present invention is directed to a process for purifying natamycin, to an all-trans polyene amphoteric macrolide, to a composition comprising said polyene amphoteric macrolide and to a process for preparing said polyene amphoteric macrolide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/267,790, filed 10 Feb. 2021, which is the National Stage entry ofInternational Application No. PCT/EP2019/071899, filed 15 Aug. 2019,which claims priority to European Patent Application No. 18189251.4,filed 16 Aug. 2018, the content of each of these applications is hereinincorporated by reference in their entirety.

BACKGROUND Field

The present invention is directed to a process for purifying natamycin,to an all-trans polyene amphoteric macrolide, to a compositioncomprising said polyene amphoteric macrolide and to a process forpreparing said polyene amphoteric macrolide.

Description of Related Art

Natamycin (structural formula (I), pimaricin, C₃₃H₄₇NO₁₃, CAS number7681-93-8,(1R,3S,5R,7R,8E,12R,14E,16E,18E,20E,22R,24S,25R,26S)-22-[(3-amino-3,6-dideoxy-β-D-mannopyranosyl)oxy]-1,3,26-trihydroxy-12-methyl-10-oxo-6,11,28-trioxatricyclo[22.3.1.0^(5,7)]octacosa-8,14,16,18,20-pentaene-25-carboxylicacid) is an all-trans epoxide polyene amphoteric macrolide antifungalused to treat fungal infections around the eye. This includes infectionsof the eyelids, conjunctiva, and cornea. Natamycin is also used in thefood industry as a preservative for dairy products like cheese, and alsoas a preservative for meat products like sausages and for fruit.

Access to naturally produced molecules in high purity is of keyimportance for multiple reasons such as optimization of atom efficiency,minimalization of waste and avoiding unwanted side effects bycontaminants. Especially for pharmaceutical applications the bar onpurity is high and natamycin is no exception to this rule.

Natamycin is produced by fermentation of the bacterium Streptomycesnatalensis after which the product usually is obtained in the form ofplate-like crystals. One of the problems associated with natamycin isrelated to its low solubility in aqueous environment and the resultingfact that aqueous formulations are in the form of suspensions. In itsoriginal form, natamycin crystals sediment rapidly, which makesnatamycin suspensions not user friendly and difficult to dispenseevenly. WO 2006/045831 describes recrystallization in aqueousenvironment by dissolving at either low or high pH followed byre-setting of the pH to neutral to yield small, needle-shaped crystals.The latter re-crystallization process is well suited for application onindustrial scale and solves the problem of unwanted rapid sedimentationof natamycin suspensions by virtue of the crystal morphology.

Further, or alternative, improvements with respect to purity can intheory be achieved by chromatographic techniques. Unfortunately, asstated above the main problem in (preparative) chromatographicpurification is the low solubility of natamycin in aqueous environment.Consequently, sufficiently high concentrations cannot be reached and asa result designing an efficient process towards further purifiednatamycin is not possible. Although chromatographic methods have beenreported in scientific literature, all are designed to detect andquantify natamycin in various samples and are not of a preparativenature. Consequently, these methods never disclose larger concentrationsof natamycin to be applied on chromatographic material nor is there anysuggestion indicating how this would be achievable. In practice, theabove documentation discloses natamycin concentrations ranging from 0.02to 1 mg/L, which even is significantly below the solubility of natamycinin water at neutral pH (around 40 mg/L). It is possible to achievehigher concentrations of dissolved natamycin however this requires theaddition of substantial amounts of organic solvents as described e.g. inWO 2004/105491, WO 95/07998 and in H. Brik in ‘Analytical Profiles ofDrug Substances’ (1981) 10, 513-561.

The same problem also prevents the isolation, preparation,identification, and analysis of compounds that are present in natamycinsamples in extremely low amounts and which were hitherto not detectedor, depending on source or production process of natamycin, perhaps noteven present. Having access to such compounds would be a desirable toolin further analyzing, helping understand and optimizing not only theproduct natamycin but where applicable also its production process.

The present invention seeks to overcome the above problems, by providinga liquid solution of natamycin in combination with a metal salt of acarboxylic acid for use in a chromatographic purification process.Furthermore, the present invention is directed at isolating andidentifying hitherto unknown trace compounds that may be present innatamycin.

SUMMARY

Throughout the present specification and the accompanying claims, thewords “comprise”, “include” and “having” and variations such as“comprises”, “comprising”, “includes” and “including” are to beinterpreted inclusively. That is, these words are intended to convey thepossible inclusion of other elements or integers not specificallyrecited, where the context allows.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to one or at least one) of the grammatical object of thearticle.

In the context of the invention the term “solution” refers to acomposition in which one component (or mixture of components) isdissolved in another component (or mixture of components). When the onecomponent (or mixture of components) is not (fully), i.e. partially,dissolved in another component (or mixture of components), thecomposition is referred to as a “suspension”. For example, a compositioncomprising 999.98 g of water and 0.02 g of natamycin (i.e. 20 ppm)wherein the natamycin is fully dissolved is referred to as a (20 ppm)solution of natamycin in water, whereas a composition comprising 999.8 gof water and 0.2 g of natamycin (i.e. 200 ppm) wherein the natamycin ispartially dissolved is referred to as a (200 ppm) suspension ofnatamycin in water. In the context of the invention, a solution isdefined as a liquid mixture which, after centrifugation for at least 10min at 3000 rpm, results in a pellet and a supernatant, the pellet afterremoval of supernatant and drying representing no more than 0.001% ofthe weight of the starting solution before centrifugation.

In a first aspect, the invention provides a process for purifyingnatamycin comprising mixing a composition comprising crude natamycin, ametal salt of a carboxylic acid and water and subjecting the resultingmixture to chromatography whereby fractions are collected and selectedfractions that comprise natamycin are combined, wherein the amount ofsaid crude natamycin is from 1 g to 100 g/kg of the total weight of saidcomposition and wherein the concentration of said metal salt of acarboxylic acid is from 0.1 mol/L to 5 mol/L.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

In the context of the present invention, purification of natamycin bychromatography is not straightforward. Although various chromatographicprocedures may be envisaged, or are known, these are developed foranalytical purposes and involve the application of samples at very lowconcentration. The latter is important to accommodate for the lowsolubility of natamycin in aqueous systems at neutral pH. Forchromatographic purification of natamycin on preparative scale thesemethods are not suitable as the maximal amounts of natamycin are too lowto isolate quantitative amounts. There are no methods known to dissolvenatamycin in high concentration at suitable pH values in aqueoussystems, i.e. systems that do not comprise components, like e.g. organicsolvents, strong acids or bases, that negatively influence thechromatographic separation or even the chromatographic material as such.The combination of natamycin with a metal salt of a carboxylic acid athigh concentrations as such is known, albeit for different purposesand/or not in the form of an aqueous solution. For example, CN 105342987discloses a gel comprising various components, including natamycin andpotassium sorbate, for the treatment of oral ulcers. Use of the samecombination, or other metal salts of carboxylic acids, for controllingthe growth of fungi is also described in EP 2749166, U.S. Pat. No.5,738,888, WO 2009/010547, and P. Onsberg et al. (Sabouradia (1978) 16,39-46), the latter combined with as much as 60% dimethyl sulfoxide.

By applying relatively high concentrations of metal salt of a carboxylicacid, like from 0.1 mol/L to 10 mol/L or from 0.5 mol/L to 5 mol/L orfrom 1 mol/L to 2.5 mol/L, natamycin is dissolved at high concentrationslike from 1 g/L to 100 g/L, or from 2 g/L to 75 g/L or from 5 g/L to 60g/L. The pH value of the solution of the invention is, measured at 20±2°C., from 6.0 to 11, often from 6.5 to 10, or from 7.0 to 9.5. At such pHvalues the solubility of natamycin in water normally is much lower, forexample, at neutral pH values this is around 0.04 g/L (40 ppm).

In an embodiment, the metal which is part of the metal salt of acarboxylic acid is an alkali metal or an alkali earth metal, examples ofwhich are calcium, lithium, magnesium, potassium, or sodium.Practically, good results are obtained when the metal is potassium orsodium.

In another embodiment, the carboxylic acid comprises from 1 to 7 carbonatoms. Examples are acetic acid, benzoic acid, citric acid, formic acid,lactic acid, propionic acid, sorbic acid but also mixtures thereof. Goodexamples are carboxylic acids with 3 carbon atoms such as lactic acidand propionic acid and carboxylic acids with 6 carbon atoms such ascitric acid and sorbic acid. The carboxylic acid may be unsaturated withone or more double bonds. The double bounds may be cis or transoriented. A good example is a carboxylic acid having two trans orienteddouble bonds such as sorbic acid. The carboxylic acid may containhydroxyl groups, such as citric acid and lactic acid. The carboxylicacid may have a single carboxyl function, but also two, three or more.

In an embodiment, the above mixing is carried out at a temperature whichis elevated for a certain period. It is found that not only dissolutionof natamycin occurs faster, but also high final concentrations ofnatamycin are obtained and often the resulting solutions displayimproved stability. Thus, a temporary increase of temperature to from30° C. to 130° C., or from 60° C. to 120° C., or from 70° C. to 110° C.may be applied for from 2 to 200 minutes, or from 3 to 100 minutes, orfrom 4 to 60 minutes.

Remarkably, the stability of natamycin in the solution obtained above ishigh and the concentration of natamycin remains at high values, alsoafter prolonged periods of time. This effect is the most pronouncedwhere the carboxylic acid is sorbic acid. Also, this effect is mostpronounced when the metal is and alkali metal such as potassium.Accordingly, the solution of the invention unexpectedly does not requirefurther auxiliary materials such as chelating agents like EDTA orantioxidants to warrant chemical stability described in the prior art.

Accordingly, the mixing of natamycin with a metal salt of a carboxylicacid results in solutions of high concentration that can beadvantageously applied in preparative chromatography, an approach thathas not been demonstrated or suggested in the art.

In another embodiment, a diol may be added to the composition comprisingnatamycin, a metal salt of a carboxylic acid and water. The diol may beadded before, during, or after the other components are mixed. The diolpreferably has a boiling point of between 125° C. and 300° C. or between150° C. and 250° C. and the amount of diol is from 50 g/kg to 950 g/kgof the total weight of the composition. It is observed that addition ofa diol to the solution of the invention resulted in further enhancementof the stability and or a further increase of solubility of natamycin.Suitable diols are dipropylene glycol, ethylene glycol, polyethyleneglycol, propylene glycol or mixtures thereof.

The ratio of metal salt of a carboxylic acid to natamycin is from 0.1(w/w) to 50 (w/w), or from 0.2 (w/w) to 45 (w/w), or from 0.5 (w/w) to25 (w/w), or from 1 (w/w) to 10 (w/w). Alternatively, on a molar basis,the ratio of metal salt of a carboxylic acid to natamycin is from 0.5(mole/mole) to 250 (mole/mole), or from 1 (mole/mole) to 100(mole/mole), or from 2.5 (mole/mole) to 50 (mole/mole), or from 5(mole/mole) to 45 (mole/mole).

An embodiment of the invention is the use of highly concentratednatamycin solutions that are subjected to chromatography. Thus, thechromatographic processes of the invention are advantageously carriedout at unprecedented large scale. For example, when the chromatographicprocesses of the invention are carried out batch-wise, the amount ofnatamycin applied to the chromatographic material may be from 1 g to 10kg per batch, or from 5 g to 5 kg per batch, or from 25 g to 1 kg perbatch. In an example it was found that high input concentrations can beachieved by dissolving the natamycin feed in high molar potassiumsorbate.

The term “preparative chromatography” relates to methods of separatingmixtures of compounds which are dissolved in the mobile phase, ofsufficient scale to isolate relevant quantities of the compound desired.Such methods are known in the art. A suitable method for preparativechromatography is, for instance, adsorption chromatography, e.g. columnchromatography. Particularly preferred separation methods are thoseknown as HPLC (High Performance Liquid Chromatography), SFC(Supercritical Fluid Chromatography), both in batch mode and incontinuous mode, e.g. SMB (Simulated Moving Bed chromatography).

As is well known by the skilled person the term “stationary phase”relates to a suitable inert carrier material on which an interactingagent is immobilized. The term “reversed phase” relates to stationaryphases in which alkyl chains are bonded to an inert carrier material. Asuitable inert carrier material is preferably macroporous, e.g. silicagel, crosslinked polystyrene, polyacrylamide or zirconia. Silica gel isparticularly preferred. Examples of “reversed phase” stationary phasesare Symmetry C18 and Atlantis C18.

The term “mobile phase” relates to a solvent or mixture of solvents inwhich the mixture of compounds to be separated is dissolved. Suitablesolvents to be used in the preparative chromatographic process accordingto the invention are the solvents that are known to be used inanalytical chromatography. In “reversed phase” liquid chromatography asa rule polar, polar protic or aprotic solvents, or mixtures thereof areused. Suitable polar solvents are for example water in combination withmethanol or acetonitrile. In supercritical chromatography, mixtures ofcarbon dioxide and polar protic solvents, e.g. methanol are preferred.

In an embodiment, columns used in preparative chromatography arevertical cylindrical tubes packed with chromatography media intended tobind the target molecules and then elute them slowly with a buffer andcollecting various fractions of the eluent. The fractions containing thetarget molecule in its purest form are then pooled to obtain a desireddegree of purification. However, the skilled person is aware thatalternative configurations are available.

In a second aspect, the invention provides(1R,3S,5E,7R,11R,13E,15E,17E,19E,21R,23S,24R,25S)-21-[(3-amino-3,6-dideoxy-β-D-mannopyranosyl)oxy]-1,3,7,25-tetrahydroxy-11-methyl-9-oxo-10,27-dioxabicyclo[21.3.1]heptacosa-5,13,15,17,19-pentaene-24-carboxylicacid of formula (II), C₃₃H₄₉NO₁₃, or a salt thereof.

It was found that by performing the process of the first aspect of theinvention the hitherto unknown compound of formula (II) can be isolatedand subsequently analyzed and identified.

In an embodiment, the invention provides a composition comprisingnatamycin and the compound of formula (II) wherein the amount of saidcompound of formula (II) is from 0.001-0.1% (w/w) relative to the amountof natamycin. Preferably, the composition is isolated, for example bymeans of lyophilization, spray-drying or other means of removing asignificant amount of water masses following the process for purifyingnatamycin of the first aspect of the invention. Consequently, thecomposition of the second aspect comprises from 0.001-10% (w/w) ofwater, or from 0.002-5% (w/w) of water, or from 0.005-3% (w/w) of water.In other words, the composition of the second aspect is a compositionobtainable by a process for purifying natamycin comprising mixing acomposition comprising crude natamycin, a metal salt of a carboxylicacid and water and subjecting the resulting mixture to chromatographywhereby fractions are collected and selected fractions that comprisenatamycin are combined, wherein the amount of said crude natamycin isfrom 1 g to 100 g/kg of the total weight of said composition and whereinthe concentration of said metal salt of a carboxylic acid is from 0.1mol/L to 5 mol/L. The composition can further provide another novelcomponent, namely(1R,3S,5R,7R,8E,12R,14Z,16E,18E,20E,22R,24S,25R,26S)-22-[(3-amino-3,6-dideoxy-β-D-mannopyranosyl)oxy]-1,3,26-trihydroxy-12-methyl-10-oxo-6,11,28-trioxatricyclo-[22.3.1.0^(5,7)]octacosa-8,14,16,18,20-pentaene-25-carboxylicacid of formula (III), C₃₃H₄₇NO₁₃, or a salt thereof. The amount of thecompound of formula (III) is from 0.001-0.1% (w/w) relative to theamount of natamycin.

In a third aspect, the invention provides a process for preparing thecompounds of the second aspect comprising subjecting natamycin or themother liquor of re-crystallized natamycin to chromatography wherebyfractions are collected and selected fractions that comprise saidcompounds are combined. Combined fractions may be concentrated toisolate the respective compounds. Concentration may be realized usingtools available to the skilled person such as evaporation,lyophilization but also crystallization or precipitation followed byfiltration.

An embodiment of the invention is the use of highly concentratednatamycin solutions that are subjected to chromatography. Thus, thechromatographic processes of the invention are advantageously carriedout at unprecedented large scale. For example, when the chromatographicprocesses of the invention are carried out batch-wise, the amount ofnatamycin applied to the chromatographic material may be from 1 g to 10kg per batch, or from 5 g to 5 kg per batch, or from 25 g to 1 kg perbatch.

An embodiment of the invention is subjecting of the mother liquor ofre-crystallized natamycin to preparative chromatography. Such a motherliquor may, for example, be obtained following re-crystallization ofnatamycin as described in WO 2006/045831. This can be dissolution ofnatamycin at high pH followed by crystallization at neutral pH ordissolution of natamycin at low pH followed by crystallization atneutral pH. The advantage of using the mother liquor of re-crystallizednatamycin in preparative chromatography is in the fact that motherliquors are generally relatively higher in impurities compared tonatamycin than the original crystals and certainly than the resultingcrystals. Consequently, the use of the mother liquor of re-crystallizednatamycin will lead to a higher yield of the desired compound followingchromatography and/or a higher purity and/or a less elaborate procedure.

In a fourth aspect the invention provides the use of the compounds orthe compositions of the second aspect in analysis of natamycincontaining samples. Designing and optimizing natamycin productionprocesses is best served when as many possible or probable side productsor contaminants or the like are known. But importantly, not only knownbut also available to the technician enabling him to consistently repeatanalytical procedures and obtain reliable results. The compounds andcompositions of the instant invention are an addition to the toolbox ofthe skilled technician enabling him to further improve and optimizeanalytical procedures and consequently elevate not only processunderstanding but also product quality to a still higher level. Theseare constant needs in modern day product development.

In an embodiment, the compounds or the compositions of the second aspectmay be used as standards and/or references in analytical methods such asHigh-Performance Liquid Chromatography, mass spectrometric analysis,Thin Layer Chromatography or NMR. Having access to larger quantitiesallows for constant availability and quality of the references andstandards. Consequently, the compound of formula (II), but also that offormula (III), is industrially applicable for various purposes rangingfrom further optimization of natamycin production to potentialantifungal activity in itself. The antifungal activity may be used invarious food applications.

The invention is further described with reference to the followingnon-limiting examples.

EXAMPLES General

HPLC in Combination with High Resolution Mass Spectrometer (LC-MS)

-   LC Column: Waters, Symmetry C18-   Mobile phase: Solvent A: 50 mM ammonium acetate buffer pH 5.8    -   Solvent B: Acetonitrile-   Injection volume: 10 μL-   Column temp.: 25° C.-   Flow rate: 1 mL/min-   MS Instrument: LTQ Orbitrap-   LC/MS: Full scan ESI positive mode

HPLC-UV Conditions for Fractionation

-   Column: Dr. Maische, NovoGROM Spherical C18, 100 Å, 250×4.6 mm, 15    μm-   Mobile phase: Solvent A: 50 mM ammonium acetate buffer pH 5.8    -   Solvent B: Acetonitrile

Gradient: Time (min.) Solvent A (%) Solvent B (%) 0.0 75 25 15.0 75 2521.0 65 35 25.0 50 50 26.0 75 25 30.0 75 25

-   Injection volume: 100 μL-   Column temp.: 25° C.-   Flow rate: 1.0 mL/min.-   Sample temp.: 15° C.-   Runtime (min.): 30 minutes

Dionex Ultimate DAD-3000 (UV Single Wavelength)

Wavelength 305 nm Bandwidth 1 nm Data collection rate 5 Hz

Dionex Ultimate DAD-3000 (UV 3D)

Wavelength 200-600 nm Bandwidth 4 nm Data collection rate 5 Hz Responsetime 1 s

The separation of compounds (I), (II), and (III) was verified using theconditions described above by means of high-resolution massspectrometry. Start collecting 20 fractions of compounds (II), (III),and natamycin (I). Collected fractions were lyophilized for isolation ofthe respective compounds and further analyzed for structure elucidation.

For reference purposes NMR spectra of natamycin (I) were recorded on aBruker Ascend 600 spectrometer. Methanol was used as chemical shiftreference (δ=3.31 ppm, 49.2 ppm). ¹H and ¹³C chemical shifts wereextracted from the ¹H-¹³C correlation (HSQC) spectrum.

Example 1 Preparation of(1R,3S,5E,7R,11R,13E,15E,17E,19E,21R,23S,24R,25S)-21-[(3-amino-3,6-dideoxy-β-D-mannopyranosyl)oxy]-1,3,7,25-tetrahydroxy-11-methyl-9-oxo-10,27-dioxabicyclo[21.3.1]heptacosa-5,13,15,17,19-pentaene-24-carboxylicacid (II)

Natamycin was produced by a culture of Streptomyces natalensis bacteriafollowing a controlled fermentation process, for example as described inWO 97/29207 or references therein. A sample of the resulting broth wasmixed with an aqueous potassium sorbate solution (3M) and the mixturewas stirred until all natamycin was dissolved. The amount of aqueouspotassium sorbate solution was such that the resulting concentration ofnatamycin was 10 g/L (w/w, 1%). In alternative, otherwise similar,experiments dissolution of natamycin was facilitated by raising thetemperature to 40±10° C. for 30±20 min followed by cooling down to 20±2°C. The resulting solution was subjected to semi-preparativeHigh-Performance Liquid Chromatography as described under General(HPLC-UV conditions for fractionation) above to isolate quantities ofcompound (II). In addition, fractions comprising a second compound offormula (III) were isolated. Samples were obtained as per the belowTable. Contents of the samples were determined with LC-UV.

RT Natamycin I II III Sample # (min) (mg/L) (mg/L) (mg/L) 1 6.627 n.a.n.a. n.a. 2 8.363 0.02 0.009 n.a. 3 9.777 0.03 0.007 n.a. 4 11.173 0.030.266 n.a. 5 13.327 0.07 0.018 n.a. 6 14.333 0.32 n.a. 0.581 7 17.5030.38 0.034 n.a. 8 20.140 134.93 0.307 0.714 9 23.377 403.18 0.465 1.694

The chemical structures were elucidated by Mass Spectrometry (MS) andNuclear Magnetic Resonance (NMR).

Example 2 Structure Elucidation of(1R,3S,5E,7R,11R,13E,15E,17E,19E,21R,23S,24R,25S)-21-[(3-amino-3,6-dideoxy-β-D-mannopyranosyl)oxy]-1,3,7,25-tetrahydroxy-11-methyl-9-oxo-10,27-dioxabicyclo[21.3.1]heptacosa-5,13,15,17,19-pentaene-24-carboxylicacid (II)

All material from the fraction comprising compound (II) obtained inExample 1 was dissolved in CD₃OD (0.6 mL). The sample presumablycontained a large amount of glycerol, introduced as a contaminant duringfreeze-drying. Also signals of free fatty acids were dominant. NMRspectra were recorded on a Bruker Ascend 700 spectrometer operating at aproton frequency of 700 MHz equipped with a TCI cryo probe and measuredwith a probe temperature of 300K with suppression of the water signal. A1D ¹H spectrum was obtained with 256 scans in 30 minutes, a COSYspectrum was recorded with 8 scans and 800 increments in F1 in 3 hours,TOCSY spectra were recorded with 8 scans and mixing times of 40 and 100ms and 800 increments in the F1 dimension in 3 hours each. A ¹H-¹³Ccorrelation (HSQC) spectrum was recorded with 288 scans and 512increments in the F1 dimension in 55 hours.

From the ¹H NMR spectrum it was clear that the signal of H9 of natamycinwas missing. Identification of the compound in this fraction waspossible by comparing the proton carbon correlation spectrum (HSQC) andthe proton-proton correlation spectrum (COSY) with those of natamycin.Overlaying the HSQC spectrum of natamycin and that of this fractionshowed that most signals had (near) identical chemical shifts with theexception of those at atom numbers 5, 6, 7, 8 and 11 of the compound offormula (II) (note that atoms 5, 6, 7, 8 and 11 in (II) are 5, 7, 8, 9and 12 respectively in (I)). With the aid of the COSY spectrum and themolecular formula given by LC/MS (natamycin+2H) the abovementionedsignals were assigned, and it was concluded that the impurity has thestructure as given in formula (II).

The characteristic doublet of natamycin (I) of H9 at 6.06 ppm hasdisappeared, and instead H8 in (II) appeared as two mutually coupledsignals at 2.28 and 2.50 ppm. These chemical shifts strongly indicate aCH₂ group. Furthermore, the characteristic double doublet of H8 innatamycin (I) has disappeared, and the proton signal of H7 in (II) wasidentified at 4.29 ppm, which is strongly indicative of a CH(OH) group.Finally, H7 and H5 in (I) were found at 3.14 and 2.82 ppm, respectively,in agreement with the epoxide moiety. In the analysis, these protonsignals, labelled H6 and H5 were both found at 5.48 ppm. Chemical shiftsof the protons and carbons that differ significantly from those innatamycin are given in the below Table. These ¹H and ¹³C chemical shiftswere extracted from the ¹H-¹³C correlation (HSQC) spectrum. Themultiplicity of the ¹H signals was not given, as all the proton signalspartially overlap with other signals.

TABLE Chemical shifts of the characteristic carbons and protons in (II)as compared to shifts predicted by ACD software version 4.04. ACD ACDACD ACD Atom nr ¹H ¹³C prediction prediction prediction prediction(IUPAC) (ppm) (ppm) ¹H trans ¹³C trans ¹H cis ¹³C cis 8 2.50/2.28 44.52.5 42 2.5 42 7 4.29 70.4 4.6 67 4.4 63 6 5.45 130.5 5.7 132 5.7 127 55.45 135.7 5.7 114 5.7 127 11 4.92 71.5 4.6 74 4.6 74

Example 3 Structure Elucidation of(1R,3S,5R,7R,8E,12R,14Z,16E,18E,20E,22R,24S,25R,26S)-22-[(3-amino-3,6-dideoxy-β-D-mannopyranosyl)oxy]-1,3,26-trihydroxy-12-methyl-10-oxo-6,11,28-trioxatricyclo[22.3.1.0^(5,7)]octacosa-8,14,16,18,20-pentaene-25-carboxylicacid (III)

All material from the fraction comprising compound (III) obtained inExample 1 was adsorbed on a Sep-Pak column. The column was rinsed withD₂O (1 mL). Next, the column was flushed with nitrogen gas. The columnwas eluted in the reversed direction with CD₃CN (1 mL). Compound (III)did not elute from the column which was subsequently eluted withCDCl₃/CD₃OD 1/1. The solution was transferred into an NMR tube, andfurther concentrated by means of a stream of nitrogen gas, until avolume of 0.65 mL was obtained. One drop of D₂O was added to improve thelinewidth in the NMR spectrum.

NMR spectra were recorded on a Bruker DRX 600 spectrometer, operating ata ¹H frequency of 600 MHz. An inverse probe equipped with gradient coilswas used. In addition to a ¹H NMR spectrum, COSY, TOCSY and HSQC spectrawere recorded. For chemical shift prediction ACD software version 4.04was used.

Comparison of the 2D NMR spectra with those of natamycin (I)demonstrated that compound (III) differs from natamycin (I) only in theconfiguration of the double bond C14-C15, being cis in compound (III)and trans in natamycin (I). The pattern of coupling constants of H14 wasin agreement with a cis-double bond on C14-C15. When the spectra ofnatamycin (I) and compound (III) were compared, it appeared that mostsignals had nearly identical chemical shifts, except those of C/H 12, 13and 14. Moreover, the coupling pattern of H14 was very different fromthe pattern seen in natamycin (I). The slightly distorted quartet of H14arose from three coupling constants of ±9 Hz, a typical value for a ciscoupling, while a trans coupling usually has a magnitude of 12-18 Hz.Furthermore, the ¹³C chemical shift of C13 has shifted 6 ppm upfield,being the expected upfield shift typical of a CH₂ adjacent to a cisdouble bond as compared to a trans double bond (ACD prediction). Thechemical shifts of the relevant protons and carbons are listed in thebelow Table. From the results it was concluded that compound (III) is anisomer of natamycin (I) with a cis double bond between C14 and C15.

TABLE Chemical shifts of the characteristic carbons and protons in (III)and in natamycin (I) as compared to shifts predicted by ACD softwareversion 4.04. ACD ACD Atom nr ¹H J ¹³C prediction prediction IUPAC (ppm)(Hz) (ppm) ¹H ¹³C Compound (III) cis cis 12 5.07 71.0 4.93 67.8 132.62/2.39 34.2 2.40 35.1 14 5.52 ±9 (3x) 127.6 5.60 127.6 Natamycin (I)trans trans 12 4.69 71.2 5.00 70.4 13 2.37/2.26 40.6 2.34/2.30 41.5 145.57 5.7, 10.2, 128.7 5.62 129.7 14.6

1. A process for purifying natamycin comprising mixing a compositioncomprising crude natamycin, a metal salt of a carboxylic acid and waterand subjecting the resulting mixture to chromatography whereby fractionsare collected and selected fractions that comprise natamycin arecombined, wherein the amount of said crude natamycin is from 1 g to 100g/kg of the total weight of said composition and wherein theconcentration of said metal salt of a carboxylic acid is from 0.1 mol/Lto 5 mol/L.
 2. The process according to claim 1 wherein said mixing iscarried out at a temperature of from 30° C. to 130° C. wherein saidtemperature is maintained for from 10 to 200 minutes.
 3. The processaccording to claim 1, wherein said metal is an alkali metal or an alkaliearth metal.
 4. The process according to claim 1, wherein saidcarboxylic acid is acetic acid, benzoic acid, citric acid, formic acid,lactic acid, propionic acid, sorbic acid, or a mixture thereof.
 5. Theprocess according to claim 4, wherein said carboxylic acid is sorbicacid.
 6. A compound of formula II comprising(1R,3S,5E,7R,11R,13E,15E,17E,19E,21R,23S,24R,25S)-21-[(3-Amino-3,6-dideoxy-β-d-mannopyranosyl)oxy]-1,3,7,25-tetrahydroxy-11-methyl-9-oxo-10,27-dioxabicyclo-[21.3.1]heptacosa-5,13,15,17,19-pentaene-24-carboxylicacid of formula (II) or a salt thereof


7. A composition comprising natamycin, water and the compound or saltthereof according to claim 6 wherein the amount of water is from0.001-10% (w/w) and wherein the amount of said compound or salt thereofaccording to claim 6 is from 0.001-0.1% (w/w) relative to the amount ofnatamycin.
 8. The composition according to claim 7 further comprising acompound of formula III or salt thereof,(1R,3S,5R,7R,8E,12R,14Z,16E,18E,20E,22R,24S,25R,26S)-22-[(3-amino-3,6-dideoxy-β-d-mannopyranosyl)oxy]-1,3,26-trihydroxy-12-methyl-10-oxo-6,11,28-trioxatricyclo[22.3.1.0^(5,7)]octacosa-8,14,16,18,20-pentaene-25-carboxylicacid wherein the amount of said compound of formula (III) or saltthereof is from 0.001-0.1% (w/w) relative to the amount of natamycin


9. A process for preparing a compound or salt thereof according to claim6 comprising subjecting natamycin or a mother liquor of re-crystallizednatamycin to chromatography whereby one or more fractions are collectedand selected fractions that comprise said compound or salt thereof arecombined.
 10. The process according to claim 9, wherein said combinedselected fractions are concentrated.
 11. The process according to claim10, wherein the fractions are concentrated by lyophilization.
 12. Theprocess according to claim 9, wherein said chromatography is carried outwith from 1 g to 10 kg of natamycin per batch.
 13. A method of analyzinga natamycin sample comprising using a compound or salt according toclaim 6 in as a standard in an analytical method of natamycin.